Computer interfaced scanning fluorescence lifetime microscope applied to directed evolution methodologies and methods for light-mediated patterning in cell selection

ABSTRACT

This invention provides a method for screening large numbers of individual cells or colonies of cells using scanning microscopy coupled with fluorescence lifetime measurement and analysis, using time-correlated single photon counting. This invention further provides an automated method for selecting cells that exhibit desired characteristics. The method uses the scanning microscope system to focus a laser beam onto a surface upon which cells are immobilized on the timescale of the procedure. The cells that are illuminated in this way are killed or their growth is inhibited. The focused laser beam is scanned across the surface and turned on and off during the scanning process such that only non-irradiated cells survive, resulting in a patterned cell growth This invention further provides a computer-controlled projection device, such as a micro-mirror array or a liquid crystal display system, which is sued to project an image onto the cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of International Application No.PCT/US01/24365, entitled SCANNING FLUORESCENCE LIFETIME MICROSCOPE:DIRECTED EVOLUTION, filed Aug. 2, 2001, which in turn claims priority toU.S. Provisional Application No. 60/222,691, filed Aug. 2, 2000,entitled COMPUTER INTERFACED SCANNING FLUORESCENCE LIFETIME MICROSCOPEAPPLIED TO DIRECTED EVOLUTION METHODOLOGIES AND METHODS FORLIGHT-MEDIATED PATTERNING IN CELL SELECTION, which are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to an apparatus andmethods for rapidly and automatically screening and selecting cellsexhibiting desirable physical traits and more specifically to a computerinterfaced scanning fluorescence microscope applied to directedevolution methodologies and methods for light-mediated patterning incell selection.

BACKGROUND OF THE INVENTION

[0003] Directed evolution is a process wherein the sequence of a gene isvaried randomly by any of a number of methods, generating a library ofmutated genes. These mutated genes are expressed and the functions ofthose gene products are assayed. A selection procedure is then appliedto select those cells containing genes that express products-withdesirable functions. These cells, and their genes, are then selectivelyamplified, and the mutagenesis, screening and selection process isrepeated until gene products with the most desirable functions areobtained.

[0004] The general scheme for directed evolution is shown in FIG. 1.First, variation is introduced into the gene in question by some type ofrandom mutagenesis and a library of sequences is introduced into anorganism (typically Escherichia coli) for expression of the alteredproteins. Next, this population of bacteria is screened for the desiredactivity and individual colonies are selected. Finally, these selectedbacteria are grown up (amplification of the selected genetic variants)and the plasmids expressing proteins with the most desirable functionaltraits are isolated. These then are used as heterogeneous templates forfurther random mutagenesis and reintroduced into the bacterium foranother round of screening and amplification. This cycle is continueduntil the desired functional characteristics are achieved.

[0005] Directed evolution has been successfully used to generate newmolecules with altered physical characteristics. For example, Doi et al.modified green fluorescent protein (GFP) to include a binding site forthe TEM1-lactamase inhibitor and then used directed evolution methods toproduce a protein molecule whose fluorescent properties changed uponbinding the target molecule. N. Doi and H. Yanagawa (1999) “Design ofgeneric biosensors based on green fluorescent proteins with allostericsites by directed evolution,” FEBS Letters 453, 305-307). Directedevolution methodologies involving fluorescent proteins are particularlyuseful as fluorescence lends itself to sensitive and relatively easy,albeit slow, visual measurement.

[0006] GFP is one of a few different proteins that, in the absence ofany externally supplied cofactor, fluoresce strongly in the visibleregion of the spectrum. Two of these proteins, GFP and a related redfluorescing protein (RFP) from reef corals, are commercially availablein the form of expressible plasmids. Tsien, R. Y. (1998) “The GreenFluorescent Protein,” Annu. Rev. Biochem. 67:509-544; Matz, V., et al.(1999) “Fluorescent proteins from nonbioluminescent Anthozoa species,”Nature Biotechnology 17: 1969-1973. Functional transgenic expression ofthese fluorescent proteins is nearly universal in both eukaryotes andprokaryotes. Both the green and red fluorescing proteins have similarstructural features, involving a beta-can fold structure enclosing achromophore that is made via a reaction between 3 consecutive aminoacids, serine, tyrosine and glycine. The quantum yield of fluorescencefrom the green fluorescent protein is near unity, while that from thered protein is apparently lower. Proteins with a variety of intermediatewavelengths have also been characterized.

[0007] Most of the directed evolution studies performed to date haveutilized visual, qualitative screening of colonies on plates followed bymanual selection of colonies that have enhanced activity in the proteinof interest. Selection of cells may be based on a number of criteria,including color, morphology, size and fluorescence, depending on theprotein of interest and the selectable marker chosen. When screeningfluorescing cells, the process typically involves exciting cells withlight and observing fluorescence from the genes or from molecules madeby or associated with the genes in the cells. This visual screeningprocess is slow and not particularly amenable to automation. As aresult, the number of cells that can be screened and selected forfurther processing is greatly limited.

[0008] Although electronic cameras have been used to record fluorescencelevels from colonies of cells, only the total relative yield of thefluorescence is recorded. This does not distinguish between fluorescenceamplitude, which depends on both the photophysical properties of thefluorophore and its concentration, and fluorescence lifetime, whichdepends only on the photophysical properties of the fluorophore. Thus,directed evolution procedures that rely on steady state measurements offluorescence select for changes that can be in either the amount of orthe chemical properties of the fluorophore, but cannot specificallyselect for changes in molecular properties independent of concentration.

[0009] Also, while the use of electronic cameras has made it possible toscreen cells more rapidly, its application has been limited by theability to manually select cells exhibiting desired traits. What isneeded, therefore, is a more sensitive, higher resolution system thatquantitates levels of fluorescence from microcolonies (colonies with adiameter of approximately 100 microns or less) or from individual cells,thus allowing cell screening on the order of millions of cells per roundof directed evolution, coupled with an automated system for selectingthe microcolonies or cells of interest.

[0010] Thus, the ability to perform directed evolution using a highresolution fluorescent assay that is sensitive, amenable to automation,and that distinguishes between fluorescence amplitude and fluorescencelifetime would be a significant asset for research as well asdiagnostics and therapeutics.

SUMMARY OF THE INVENTION

[0011] This invention relates to a method for screening large numbers ofindividual cells or microcolonies based on fluorescence lifetime offluorescent markers present in the cells. This invention involves:

[0012] providing a substrate with multiple locations, at least some ofwhich contain one or more cells containing a fluorescent marker;

[0013] directing a light beam onto each location, thereby causing thefluorescent marker to emit fluorescent light;

[0014] automatically detecting the fluorescent light;

[0015] automatically measuring and recording the lifetime of thefluorescent light; and

[0016] correlating the lifetime of the fluorescent light with thelocation containing the cell with the fluorescent marker emitting thefluorescent light.

[0017] This invention further relates to a method for generating ahigh-resolution image map of cell fluorescence lifetime and using theimage map to select cells exhibiting desired fluorescent properties.

[0018] This invention further relates to a method for automaticallyselecting cells exhibiting desired characteristics of imagableproperties, such as fluorescence, color, morphology, or any othercharacteristic that may be detected and recorded, by selectively killingthose cells not exhibiting the desired characteristics. In oneembodiment, this involves:

[0019] providing a substrate with multiple locations, at least some ofwhich contain one or more cells expressing an imagable property;

[0020] detecting and recording the imagable property;

[0021] identifying and recording locations containing cells expressing adesired characteristic of the imagable property and locations notcontaining cells expressing the desired characteristic of the imagableproperty; and

[0022] scanning lethal irradiation across the substrate through a highspeed shutter and through an objective, wherein the shutter is open onlywhen the objective is positioned over locations not containing cellsexpressing the desired characteristic of the imagable property tothereby kill the cells in such locations.

[0023] In an alternative embodiment, the invention involves:

[0024] providing a substrate with multiple locations, at least some ofwhich contain one or more cells expressing an imagable property;

[0025] detecting and recording the imagable property;

[0026] identifying and recording locations containing cells expressing adesired characteristic of the imagable property and locations notcontaining cells expressing the desired characteristic of the imagableproperty; and

[0027] projecting lethal irradiation only onto those locations notcontaining cells expressing the desired characteristic of the imagableproperty to thereby selectively kill those cells.

[0028] In both cases, light that is not in and of itself lethal can beused in place of lethal radiation if the cells are first treated with asensitizing agent or are induced to synthesize endogenous sensitizingagents.

[0029] This invention further provides for an apparatus for theautomated screening and selection of cells based on fluorescenceproperties.

[0030] This invention may be used with both prokaryotic and eukaryoticcells. This invention is useful in directed evolution methodologies butalso may be used to screen and select cells in situ.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1 is a schematic diagram illustrating the general process fordirected evolution.

[0032]FIG. 2 is a schematic diagram of one embodiment of a fluorescencelifetime imaging system.

[0033]FIG. 3 is a schematic diagram of a four channel fluorescencelifetime imaging system.

[0034]FIG. 4 is a schematic diagram illustrating the use of a confocalmicroscope with a scanning stage to inspect and evaluate individualcells for fluorescence lifetimes and amplitudes, followed by selectivekilling of undesirable cells by an intense burst of ultraviolet lightfrom an electro-optically shuttered argon laser.

[0035]FIG. 5 is a schematic diagram illustrating the use of a CCD camerato screen for fluorescence from cells and a digital light processor tospecifically irradiate cells with ultraviolet light to kill individualcells that do not exhibit desired characteristics.

[0036]FIG. 6 is a flowchart summarizing the use of the computerinterfaced scanning fluorescence microscope in directed evolutionmethodologies.

[0037]FIG. 7 is a flowchart summarizing the use of light mediatedpatterning in cell selection using imaging of lethal irradiation indirected evolution methodologies.

[0038]FIG. 8 is a graph illustrating time-correlated fluorescent signalsfrom an individual E. coli cell expressing red fluorescent protein.

[0039]FIG. 9 is an illustration of one example of an optically patternedcell growth. FIG. 9A is a drawing of an image projected onto E. coligrown in the presence of a cationic porphyrin and plated onto anLB-plate, while FIG. 9B is a photograph of the pattern of cell growthresulting after the plate is exposed to visible light.

DETAILED DESCRIPTION OF THE INVENTION

[0040] This invention provides a method for screening large numbers ofindividual cells or colonies of cells using scanning microscopy coupledwith fluorescence lifetime measurement and analysis, usingtime-correlated single photon counting. Both the imaging of thefluorescence lifetime data from cells and/or colonies on a surface andthe analysis of this data are controlled and performed in an automatedand rapid manner using a computer. This screening method can then beused with either light-mediated patterned cell growth methodologies, asfurther provided by this invention, or mechanical methods to selectindividual cells or colonies based on their fluorescent properties.

[0041] This invention provides two distinct improvements over currentmethods for screening cells. First, automated scanning of thefluorescent properties of cells or colonies enables a large number ofcolonies to be screened rapidly and automatically. In the practice ofthis invention, a slide containing millions of cells can be examined inminutes. Second, the method allows one to determine independently thelifetime and the amplitude of the fluorescence. Current methods ofscreening involve either a manual or automated survey of totalfluorescence, which depends on both the lifetime and the amplitude ofthe fluorescence. By distinguishing between lifetime and amplitude, onecan determine whether changes in fluorescence are due in changes innumbers of fluorophores or changes in the excited state lifetime (i.e.,the chemical properties) of the fluorophores.

[0042] The invention further provides an automated method for selectingcells that exhibit desired characteristics. In one embodiment, thismethod utilizes a computer-controlled scanning microscope system tofocus a laser beam onto a surface upon which cells immobilized on thetimescale of the procedure. The cells that are illuminated in this wayare killed or their growth is inhibited. The focused laser beam isscanned across the surface and turned on and off during the scanningprocess such that only non-irradiated cells survive, resulting in apatterned growth of cells. Alternatively, other scanning systems, suchas acousto-optical scanners, scanning mirror systems, or other scanningsystems known in the art can be used.

[0043] In an alternative embodiment of this patterned growth cellselection method, a computer-controlled projection device, such as amicro-mirror array or a liquid crystal display system, is used toproject an image onto the cells. Cells onto which this image isprojected are killed or their growth is inhibited, again resulting in apatterned growth of cells. As used herein, projection can beaccomplished by directing a specific image onto a substrate, byproviding a mask to thereby cover portions of the substrate not to beirradiated, or by other methods known in the art.

[0044] In both embodiments, inhibition of cell growth occurs either bythe use of light wavelengths that are themselves lethal to the cells,such as ultraviolet light, or via the use sensitizing chemicals thatabsorb light at particular wavelengths and generate lethal damage to thecell.

[0045] By employing this invention, cells can be selected with highspatial resolution, and large numbers of cells can be processed.Importantly, this cell selection can be done strictly based on function,as manifested in some detectable property of the cell such asfluorescence or absorbance. This is in contrast with other highthroughput selection procedures that utilize large numbers of cells, butrequire that the selected trait confer a significant growth advantage.This process is preferably coupled with high throughput imaging of cellfluorescence using either a sensitive charge couple device based camera(CCD) camera or a scanning microscope.

[0046] This invention permits selection of desirable cells in directedevolution techniques, since cells can be selected with great resolutionat sub-visual sizes, allowing a vast number of cells to be processed atonce, without the need for antibiotic resistance markers or growth onselective media lacking required nutrients. This invention can also beused in color-based assays for transformation of bacterial cells withplasmid DNA, obviating the need for antibiotic resistance. Further, cellpatterning can be used with essentially any cell type, including yeastand mammalian cells, using appropriately selected or modified chemicalsensitizers.

[0047] Spatially Imaged Fluorescence Lifetime Detection Device

[0048] The spatially imaged fluorescence lifetime detection devicecomprises a scanning microscope system with a nanopositioning ormicropositioning stage, or a laser scanning system, modified by theinclusion of a pulsed excitation source, a photon counting detector andappropriate time correlation electronics. In one embodiment, a confocalmicroscope is used, although other microscope systems may also be used.The positioning capability can be in either two or three dimensions, andallows computer controlled movement system that can position the focalpoint of a beam on a sample with submicron accuracy. Such positioningstages or scanning systems are commercially available from, for example,Mad City Labs (Madison, Wis.; Nanoh100-xy), PI (Physics Instruments,Germany) or Brimrose Corporation of America (Baltimore, Md.).Alternatively, the stage may be kept stationary while the beam is movedrelative to the stage.

[0049] The pulsed excitation source can be any laser or light sourcewith a high repetition rate and a short pulse width, generating pulsesat greater than 10 kHz. In one embodiment, an actively mode-locked NdYAGlaser is used, generating pulses at 80 MHz, which, after compression,are 5 ps in duration. The wavelength used to excite the sample variesaccording to the sample. In another specific embodiment, an ultrafasttitanium sapphire oscillator is used, pumped by a continuous lasersource such as a diode-pumped NdYAG laser. The oscillator producespulses of about 100 femtosecond duration at a repetition rate of 80 MHz.

[0050] The photon counting device may be any detector capable ofdetecting and counting photons, generating electrical pulses for eachphoton detected. In one embodiment, an avalanche photodiode is used.Alternatively, a photomultiplier tube is employed. Such devices are wellknown in the art.

[0051] The time correlation electronics is any device that can receiveinformation both from the photon counting device and from the laser, orfrom a fast photodiode associated with the laser, and record time in twodimensions. Preferably, the device uses time correlated single photoncounting (TCSPC) to determine the time between a laser pulse and theresulting photon emission (i.e., the excited state lifetime of themolecule giving rise to the photon, generally in the nanoseconds timeframe) and it records the time at which the photon arrives, in the labtime frame, typically with microsecond to millisecond accuracy. Suchtime correlation electronics are commercially available from, forexample, Becker & Hickl (Berlin, Germany) or PicoQuant (Berlin,Germany).

[0052] In the practice of the invention, a beam from the high repetitionrate pulsed laser is passed into the microscope, reflected from adichroic mirror, and used to excite a sample. Preferably, the samplesits on a 3-D translation/positioning stage or the laser position iscontrolled by a scanning device such as a rotating mirror or anacousto-optic scanner (these devices will be collectively referred to as“positioners”) and its position relative to the focused laser beam iscontrolled by the computer, thus allowing scanning of the sample. Thesample consists of single cells or colonies of cells sitting on, orembedded in, a solid substrate so that their positions do not vary overthe period of time required to obtain the image. The cells may be eitherprokaryotic or eukaryotic, with at least some portion of the cellsexhibiting fluorescence, or other imagable property, when excited.

[0053] Upon excitation, the sample emits a fluorescent signal thatpasses through various optical elements. In one embodiment, thefluorescence passes through the dichroic mirror, as the fluorescence isat a wavelength that is not reflected by the dichroic mirror. Eachphoton emitted by the sample is counted at the detector and the time ofarrival of each emitted photon relative to the laser pulse iscorrelated, stored and analyzed on the computer.

[0054]FIG. 2 shows one embodiment of the spatially imaged fluorescencelifetime detection system 10. A high frequency (greater than 10 kHz)pulsed laser system 12 is used as the excitation source. The laser emitsa light beam 14, which is directed via the use of a mirror 16 to adichroic mirror 18. The laser is connected to a 2-D TCSPC board 20,which receives an input from the laser that marks the time at which thelaser pulse was initiated.

[0055] The dichroic mirror 18 reflects the laser light beam 14 into themicroscope system 22, where it is directed via additional mirror(s) 24to the objective lens 26. This lens system focuses the beam onto thesample 28. The sample is attached to a computer-controlled positioningstage 30.

[0056] The laser beam excites molecules within at least some of thecells on the stage, causing them to emit light as fluorescence. Some ofthis fluorescence 32 is captured by the objective lens 26 and passedback into the microscope along the same path through which the laserlight beam 14 entered. The fluorescence is reflected from mirror 24 tothe dichroic mirror 18, where the fluorescent light passes through, asthe dichroic mirror is selected to reflect light at the wavelength ofthe laser light beam but transmit light at the wavelength of thefluorescent light. The fluorescent light 32 then passes through a filter34 to remove any remaining laser light while efficiently passing lightin the wavelength region of the fluorescence and, optionally, through aconfocal pinhole 36 (typically on the order of 50 to 150 microns indiameter and translatable along the axis of the laser beam) to betterdefine the volume of sample being probed.

[0057] The fluorescent light is detected by an avalanche photodiode 38,which generates electrical pulses for each photon of fluorescent lightit detects. These pulses are transmitted to the TCSPC board 20. TheTCSPC board records the time at which the photon arrived and uses timecorrelated single photon counting to determine the time between thelaser pulse and the photon emission. This information is transmitted toa computer 40, where it is stored and analyzed. Optionally, computer 40is interfaced with the positioning stage 30.

[0058] An alternative embodiment of the spatially imaged fluorescencelifetime detection device is shown in FIG. 3. In this embodiment, a fourchannel system records not only the excited state lifetime of eachfluorophore that gives rise to each photon detected, but also recordsthe polarization of the photon and wavelength region in which it wasemitted. This additional information can also be used to determine whichcells or colonies exhibit the most desirable characteristics; forexample, those cells containing the most desirable gene products in thedirected evolution process.

[0059] In this embodiment, the pulsed laser 42 emits a light beam 44,which is directed via the use of a mirror 46 to a dichroic mirror 48.The laser is connected to a 2-D TCSPC board 50, which receives an inputfrom the laser that marks the time at which the laser pulse wasinitiated.

[0060] The dichroic mirror 48 reflects the laser light beam 44 into themicroscope system 52, where it is directed via additional mirror(s) 54to the objective lens 56. This lens system focuses the light beam ontothe sample 58. The sample is attached to a 2-D positioning stage 60controlled by a computer (not shown).

[0061] As the laser beam excites molecules within at least some of thecells on the stage, fluorescent light 62 is emitted, some of which iscaptured by the objective lens 56 and passed back into the microscopealong the same path through which the laser light beam 44 entered. Uponreaching the dichroic mirror 48, the fluorescent light passes throughthe dichroic mirror, through a filter 63 and, optionally, through aconfocal pinhole 64.

[0062] The fluorescent light then enters a polarizer 66, emerging fromthe polarizer in two perpendicular planes 68, 70 as polarized light,each of which enters a wavelength separator 72, 74. Polarized lightpassing through the wavelength separator is again split into two pathsof light, each of which is detected by an avalanche photodiode 76, 78,80, 82. The avalanche photodiode generates electrical pulses for eachphoton of fluorescent light it detects. These pulses are transmittedthrough multiplexing electronics 84 to the TCSPC board 50. Themultiplexing electronics comprise a circuit which adds a differentperiod of delay time to the pulses arriving from different channels(Becker & Hickl, Berlin, Germany). In this way the TCSPC board is ableto differentiate between the signals from the four different detectors.The TCSPC board records the wavelength region and polarization of eachphoton, in addition to the lifetime of the excited state that gave riseto the photon. These attributes are all be recorded along with thearrival time of each photon in the lab time frame with a millisecondresolution. This information is transmitted to a computer 86, where itis stored and analyzed.

[0063] In the embodiments illustrated in FIGS. 2 and 3, a scanningfluorescent microscope is used to image the fluorescence from cells,which is then used to determine which regions of the surface are to beilluminated with lethal irradiation. Various other methods for imagingcells can also be used. For example, a charge couple device based camera(CCD camera) may be used. It is also possible to monitor absorbance in aspatially resolved fashion or to use a scanning probe microscope togenerate an image of the morphology, electrical characteristics, surfaceproperties, etc., of cells. Any imaging system with sufficient spatialresolution to resolve the features important in identifying cells withdesired properties may be employed.

[0064] Light Mediated Patterning in Cell Selection

[0065] The lifetime image determined by correlating the excited statelifetime measured by the spatially imaged fluorescence lifetimedetection system with the position of the positioner at the time of themeasurement within the lab time frame can be used to determine which ofthe cells or colonies in the sample have the desired characteristics.Then, any of several computer-controlled methods for rapidly selectingindividual cells or colonies can be employed to either removespecifically the cells of interest (positive selection) or to kill cellsthat do not have the desired qualities (negative selection). Forexample, any of several automated mechanical methods for picking cellcolonies and moving them to a clean substrate can be used. Whateverselection method is used, the lifetime image of the cells or colonies isstored on a computer and the computer can be then used to automaticallydecide which cells should be selected, using this information toinitiate an automated procedure for cell selection.

[0066] The present invention provides methods for selecting cells basedon patterned cell growth. This method employs a negative selectionstrategy, in which cells identified not to exhibit the desiredcharacteristic are selectively killed using a scanning laser.Alternatively, a light “image” is projected onto the sample that killsthe unwanted cells.

[0067] In one embodiment, fluorescence from cells on a surface isrecorded by a scanning fluorescence microscope capable of recording boththe fluorescence amplitude and its lifetime, via the use of singlephoton counting technology, as described above. The image thus obtainedof the fluorescence on the surface is used to determine which cells orcolonies exhibit the desired fluorescence characteristics. Thisinformation is processed and a new image (the “kill image”) is generatedby the computer. This kill image is designed to irradiate theundesirable cells (that is, those not exhibiting the desiredfluorescence characteristics) under conditions that are lethal to thosecells, leaving the cells with desirable fluorescence properties tocontinue growing. The kill image is projected by scanning a UV laseracross the surface of the plate, using a fast shutter (typically anacousto-optic modulator) to determine at what positions lethalirradiation occurs. One UV laser suitable for use in this invention isan argon ion laser.

[0068]FIG. 4 shows an example of the use of a UV laser with a scanningfluorescence microscope to select cells based on their fluorescentproperties. In FIG. 4, a mode-locked pulsed laser system 88 is used asan excitation source. The laser emits a light beam 90, which is directedto a dichroic mirror 92. The laser is connected to a 2-D TCSPC board(not shown), which receives an input from the laser that marks the timeat which the laser pulse was initiated.

[0069] The light beam is reflected into a microscope system by thedichroic mirror 92, into the objective lens 94. This lens system focusesthe light beam onto a sample of cells located on the surface of a plate96 positioned on a translation or positioning stage 98, causing someportion of the cells to emit fluorescent light 100. Some of thisfluorescence is captured by the objective lens and passed back into themicroscope along the same path through which the laser light entered.Upon reaching the dichroic mirror 92, the fluorescence passes through asthe dichroic mirror is designed to reflect light at the wavelength ofthe laser but transmit light at the wavelength of the fluorescence.Optionally, the fluorescence is then passed through a collimating lens102 and a confocal pinhole (not shown).

[0070] The cell fluorescence is imaged by scanning the stage, detectingthe fluorescence with a detection system 104, as shown in detail in FIG.2, and recording both the amplitude and the lifetime of the emission ateach point using time correlated single photon counting techniques. Theimage thus generated is stored and analyzed in a computer (not shown)and used to determine which cells or colonies on the surface shouldreceive lethal irradiation from the UV laser 106. A computer controlsboth the position of the stage 98 and the shutter 108 in conjunction,such that UV light 110 from the UV laser is directed via mirrors 92, 112to the sample, specifically irradiating the undesirable cells with UVlight, thus killing them. In this figure, mirror 112 is a dichroicmirror that reflects UV light, while transmitting light at thewavelength of the pulsed laser. Other configurations are possible.Importantly, the divergence properties of the UV laser are optimized sothat its focal point is in the same position along the axisperpendicular to the sample surface as the longer wavelength measuringbeam is.

[0071] It is also possible to use a visible laser beam in conjunctionwith the scanning laser approach to patterning cell growth if the cellsare first sensitized to visible light by any of a number of meansdescribed below. This has the advantage that the same laser that is usedto monitor the fluorescence from the cells can be used to kill the cellssimply by increasing the light intensity to a lethal level.

[0072] It is also possible to use an ultrafast laser pulse (on the orderof a few hundred femtosecond duration) as both the excitation source forperforming the time correlated single photon counting measurements ofexcited state lifetimes and as the source of light to directly kill theunwanted cells even without the use of sensitizers. Both processes canbe performed by multiphoton excitation. The very high peak intensity ofshort pulses make it possible for the fluorophore, such as GFP, toabsorb two photons of near infrared light and then to fluoresce in theusual visible region.

[0073] This has several advantages. First, since the excitation is inthe near infrared, it is very well separated spectrally from thefluorescence. This decreases the background due to scattering of variouskinds as well as other fluorescent materials. Second, because theprocess depends on multiple photons, the volume of material where thephoton density is high enough to cause the multiphoton absorption issmaller, increasing the spatial resolution of the technique. Finally, byusing near infrared or even infrared light as the source of photons forthe multiphoton excitation, it is possible to excite fluorophore incells below of surface of a sample allowing three dimensional mapping ofthe fluorescence, as long wavelength light will penetrate more deeplythan visible or UV light. This deep probing occurs without excitingfluorophore in the cells above, because the intensity of light will onlybe great enough as the focal point of the beam to perform themultiphoton excitation.

[0074] The same procedure can be used to kill cells by producing thesame transitions in DNA and protein molecules that make UV lightabsorption lethal, by using multiple photons of longer wavelength light.This can be done effectively by simply increasing the intensity of thelaser beam when focused on the cell or cells to be killed. Multiplephotons will then excite the same transitions in DNA and protein that UVlight does and kill the cells. The great advantage here is that a singlebeam of light can be used both to probe and to kill the cells. As notedabove for multiphoton excitation of fluorescence, multiphoton excitationof bactericidal transitions in DNA and protein molecules in the cell canbe performed with higher spatial resolution that can be done with UVlight. In addition, the near infrared or infrared laser pulses canpenetrate the surface allowing for three dimensional killing of cells.Because the unfocused light does not have the intensity to causemultiphoton excitation of lethal transitions, the killing will onlyoccur at the focal point of the laser and not in the cells above.

[0075] The intensity and wavelength of the laser beam used formultiphoton excitation screening and cell selection depends both on thespecific fluorophores being used and on the geometric constraints of thesample. The wavelength used for screening need be a multiple of theabsorbance wavelength preferred for the fluorophore to be excited. Thepower level of the laser is also dependent on the nature of thefluorophore, the concentration of the fluorophore and the size of theregion to be excited at any given time. Generally, the minimum laserintensity required to obtain a substantial signal from the fluorophoreshould be used, and this can be determined by performing test scans withincreasing light levels. For killing cells in particular patterns, thewavelength and intensity depends on the mechanism of killing employed.For example, if a specific sensitizer is used, the wavelength of thelaser need be a multiple of the absorbance wavelength of the sensitizer.The appropriate power again be determined by increasing the laser intest scans until cell death is regularly achieved. If no sensitizer isused, multiphoton excitation of DNA and/or protein molecules in the cellare possible by picking an excitation wavelength that is a multiple of awavelength in the absorbance range of these molecules (190-290 nm). Theintensity required will depend on the multiphoton absorption crosssection and the cell type. Again this can be determined empirically byincreasing the intensity in a test case until high resolution cell deathis achieved. Multiphoton beams have previously been used for“nanosurgery” at the subcellular level (Konig, 2000, J. of Microscopy,vol. 200, 83-104).

[0076] In an alternative method of light mediated patterning in cellselection, fluorescence from cells on a surface is recorded by a CCDcamera. The image of the fluorescence on the surface is used todetermine which cells or colonies contain the desired fluorescencecharacteristics (i.e., which cells are expressing biomolecules that havethe desired traits). This information is processed and a kill image isgenerated by the computer. This image is designed to irradiate the cellsthat are undesirable under conditions which will prove lethal to thosecells and leave the cells with desirable fluorescence properties tocontinue growing. In one embodiment, the kill image is the inverse ofthe fluorescence image. Thus, when the kill image is projected onto thecells, the cells with the highest fluorescence receive the leastradiation (and thus continue growing) while cells with the lowestfluorescence receive the greatest dose of radiation (and are thuskilled). Alternatively, the new image can be designed to irradiate allcells with fluorescent activity below a predetermined threshold. Otherkill image configurations are apparent.

[0077] The kill image is projected using a computer-controlled imagingsystem such as micro-mirror array chips (digital light processors orDLPs) or liquid crystal projection units that are commonly used forprojecting computer generated images on screens (available from TexasInstruments, Dallas, Tex. or InFocus Corporation, Wilsonville, Oreg.).These projection systems must be significantly modified for thispurpose. The lamp is selected to emit light at a wavelength or range ofwavelengths suitable for killing selected cells. Also, the imagingoptics must be selected both to be appropriate for the size of the imageto be generated and for the wavelength region of light used. Finally,appropriate filters must be used to select the desired wavelengthregions of light. In particular, a high quality lens system with lowoptical aberrations is used such that the inherent resolution of theinstrument is maintained when the image is reduced to the size of thetarget.

[0078] In one embodiment, the computer storing the kill image isconnected to an InFocus® model projector, which uses the video outputfrom a computer to display an image onto a screen (InFocus Corporation,Wilsonville, Oreg.). The focal optics of the projector are replaced by a50 mm Nikon® lens area, so that the output can be focused on the cellswith image features having the proper size and alignment (Nikon, Inc.,New York, N.Y.). Other lens systems can also be used, depending on thesize of the target area. The projector uses an aluminum micro-mirrorarray that is electronically controlled. Suitable chip dimensions are1024×786 pixels, although other dimensions may be used depending on thedesired resolution. When focused on an agar plate containing cells, theimage size is approximately 11 cm by 8.5 cm, producing a pixel size ofapproximately 0.07 mm/pixel. Such an optical arrangement allowsselective imaging and killing in a library of 100 micron coloniescontaining several hundred thousand members.

[0079]FIG. 5 shows a CCD camera being used to image the fluorescencefrom cells. This information is then used by the computer to generatethe kill image of lethal irradiation projected onto the plate ofbacteria. In this embodiment, the cells to be screened and selected aregrown in a plate 114 on agar or other solid substrate, preferably whichsupports growth of the cells. The plate is supported on atransilluminator 116, with a filter 118 between the plate and thetransilluminator. The filter 118 is selected to permit a wavelength orwavelengths of light suitable for exciting fluorescence of the cells topass from the transilluminator 116 to the cells on the plate 114, butnot wavelengths that would interfere with the detection of fluorescencefrom the cells.

[0080] Fluorescence 120 emitted from the excited cells is reflected froma mirror 122, through a filter 124 and a lens 126 into a CCD camera 128.The fluorescence image detected by the CCD camera is stored in acomputer (not shown) and is used to generate the kill image designed toselectively kill cells not exhibiting the desired traits. An ultravioletlight source 130 emits UV light 132 into a digital light processor 134,which projects the UV light image through a lens 136 and onto a dichroicmirror 138, selected to allow the fluorescence used for imaging by theCCD camera to pass through and UV light to be reflected. The dichroicmirror 138 reflects the UV light image onto the cells in the plate 114,selectively killing some portion of the cells.

[0081] Various other methods for imaging cells could also be used.Alternatively, one could use a scanning fluorescence microscope (oneexample is a scanning microscope capable of determining the lifetime ofthe fluorescence, its polarization and its wavelength region, as isdiscussed above with reference to FIG. 3). It is also possible tomonitor absorbance in a spatially resolved fashion or to use a scanningprobe microscope to generate an image of the morphology, electricalcharacteristics, surface properties, etc. of cells. Any imaging systemthat works with high enough spatial resolution to resolve the featuresimportant in determining which cells have the most advantageousproperties for the directed evolution project of interest would work.

[0082] Both of the above embodiments of a system for automated cellselection use UV light as the source of lethal irradiation. The use ofUV light is both a simple and an efficient means to kill cells. However,alternative methods using visible or near infrared light sources arealso available. When using visible or near infrared light, a sensitizingagent is first applied to the cells that will absorb the light, usingthe light energy to either directly damage the cell or to generate achemical species that damages the cells. Alternatively, cells may bechemically induced to produce endogenous photosensitizers.

[0083] One example of a sensitizing agent is an intercalating or DNAbinding dye, such as ethidium bromide, thiazole orange, etc. These dyesassociate directly with DNA and, upon light absorption, can cut ordamage the cell DNA, causing cell death. Alternatively, singlet oxygengenerating molecules, such as porphyrins, certain cyanine dyes and thelike may be used. In their excited state, these molecules can interactwith molecular oxygen (normally in a triplet form) to generate thehighly reactive singlet oxygen species. Singlet oxygen reacts with mostorganic compounds, often destroying their normal function in theprocess. If enough damage is done, the cell dies.

[0084] Porphyrins are strong absorbers of visible light, and many formlong lived triplet states upon excitation with visible light. Since theamount of damage done to the cells depends on the amount of lightabsorbed by porphyrins in the cell, which, in turn, is a function of theamount of porphyrin in the cell and the amount of incident light, cellsin relatively dark areas have a much higher chance of survival thancells in relatively bright areas of the image. The more porphyrin thereis in a cell, and the more light incident on that area of the plate, themore damage will be done, and thus the chance of cell survival will beless.

[0085] For gram-negative bacteria, cationic porphyrins have been foundto be the most efficient exogenous porphyrin photosensitizers. Chemicalsensitizers for a large variety of cell types, including eukaryoticcells, are well known in the art. For example, several chemicalphotosensitizers are described in the photodynamic therapy literature,in which chemicals that are taken up more rapidly by cancer cells or bypathogens relative to normal cells are used to sensitize these cells todestruction by light.

[0086] Two chemical sensitizers effective for use with E. coli, a gramnegative bacteria, are tetra(4N-methylpyridyl) porphine and tetra(4N,N,N-trimethyl-anilinium) porphine. Greater than 99.99% of E. coli cellsare killed by incubation with these porphyrins at 10 ug/mL for 5minutes, followed by irradiation at 6 mW/cm² for 20 minutes. Bycontrast, the anionic porphyrin tetra (4-sulphonatophenyl) porphineshows no photoinactivation under the same conditions. Merchat et al.(1996) Journal of Photochemistry and Photobiology B: Biology 32:153-157.

[0087] Alternatively, E. coli can be induced to synthesize endogenousporphyrins precursors by incubating the cells with d-aminolaevulinicacid at 5-9 mM for 15 minutes. Szocs, et al. (1999) Journal ofPhotochemistry and Photobiology B: Biology 50: 8-17. Followinginduction, 99.4% inactivation of E. coli is seen after 90 minutes ofirradiation at 0.08 W/cm².

[0088] Use of Computer Interfaced Scanning Fluorescence Microscope inCell Screening and Selection in Directed Evolution Methodologies

[0089] As is apparent from the above discussion, the computer interfacedscanning fluorescence microscope is useful in directed evolutionmethodologies where fluorescence, or other imagable characteristic, isused as an indicator of protein function to screen and select cells fordesired functional characteristics. An overview of this method is shownin FIG. 6.

[0090] First, the sample of the cells to be screened is placed on apositioning stage and positioned under the objective of thecomputer-interfaced scanning microscope. The computer moves thepositioning stage, recording the position of the stage relative to theobjective in lab frame. At the same time, the cells are excited by alight source, such as a mode-locked laser, through the objective of thescanning microscope. Some portion of the resulting cell fluorescencepasses through the objective and is directed to an avalanche photodiodeor photomultiplier tube, interfaced with a 2-D TCSPC board and acomputer, where the fluorescence lifetime and amplitude and is measuredand recorded. The computer is also interfaced with the laser or otherlight source and records and stores the time at which each laser pulseis initiated.

[0091] The fluorescence data is correlated with the position of thepositioning stage (and thus the position of the sample), generating ahigh-resolution image map of individual cell (or microcolony) positionsbased on the fluorescence lifetime and amplitude data.

[0092] This high-resolution map image is stored for use in identifyingindividual cells or microcolonies expressing desirable functionaltraits, as measured by fluorescence lifetime and amplitude. These cellsor microcolonies are then selected for future rounds of directedevolution.

[0093] Alternatively, the high-resolution map is used to generate ahigh-resolution kill image map of cells or colonies not expressingdesirable functional traits, as measured by fluorescence data. Usingthis kill image map, a UV laser is directed through a fast shutter andinto the objective of the scanning microscope, where it is scannedacross the sample of cells. Both the shutter and the nanopositioningstage are controlled by the computer such that the shutter is open whenundesirable cells or microcolonies are positioned under the objective,killing those cells, while the shutter is closed when desirable cells ormicrocolonies are positioned under the objective.

[0094] As is apparent, this method selectively and automatically killsundesirable cells (or microcolonies) one cell (or microcolony) at atime, greatly increasing the number of cells which can be screened andselected during directed evolution.

[0095] Use of Light Mediated Patterning Using Imaging of LethalIrradiation in Directed Evolution Methodologies

[0096]FIG. 7 shows an overview of the use of light mediated patterningusing imaging of lethal irradiation in directed evolution methodologieswhere fluorescence, or other imagable characteristic, is used as anindicator of protein function, cell morphology or activity of cellularcomponents. In this method, a high resolution kill map is projected ontoa sample, selectively killing large populations of undesirable cellswhile permitting desirable cells to continue growing.

[0097] First, a sample of cells to be screened for fluorescence isexcited by a light source such as a transilluminator, causing someportion of the cells to fluoresce. This fluorescence is detected andrecorded by an electronic camera, such as a CCD camera, interfaced witha computer. The fluorescence data is used to create a high-resolutionmap correlating fluorescence with cell (or microcolony) position withinthe sample.

[0098] Next, the fluorescence image is used to generate ahigh-resolution kill map of cells (or microcolonies) not expressingdesirable functional characteristics. Lethal irradiation, such as UVlight, is projected onto the sample in the form of the kill image.Alternatively, the sample is first treated with a photosensitizer, orendogenous photosensitizers are induced, and a wavelength of lightabsorbed by the photosensitizer is projected onto the cells in the formof the kill image. The projection of the kill image may be controlled bya digital light processor interfaced with the computer storing the killimage. In any case, the irradiation selectively kills the cells on whichthe kill image falls, leaving the nonirradiated cells to continuegrowing.

[0099] Because the kill image is a high resolution map based onfluorescence data correlated to desirable functional characteristics,this method of light mediated patterning is a rapid and efficient way tosimultaneously select large numbers of cells or microcolonies forfurther directed evolution studies.

[0100] As is apparent, the detection technique used to generate the highresolution map may by varied depending on the imagable property ofinterest. So long as the property of interest, whether it is cellmorphology, calorimetric reactions or the like, can be imaged, a highresolution map can be generated for use in patterned cell selection asdescribed for fluorescing cells.

[0101] The cell screening and selection methods described above are notlimited to use with bacterial cells. The methodologies of this inventionhave potential applications for eukaryotic cells as well. For example,patterned cell selection can be used for the selection of yeast cells,which are often used in various techniques in which libraries of genesequences are generated and specific colonies are selected. Patternedcell selection could also be used in the selection of mammalian cellsfor similar reasons.

[0102] In addition, patterned cell selection could have directapplication in the field of photodynamic therapy presently used fortreatment of certain types of cancer. Porphyrins selectively accumulatein certain types of cancer cells, causing the cancer cells to fluoresce.Presently, light is used to illuminate all cells in the vicinity of thecancer cells. The cancer cells, due to their higher concentration ofporphyrin, are killed faster than the normal cells, but many normalcells die as well.

[0103] By using the fluorescence from the porphyrin or from a specificbinding fluorophore in the cancer cells to map the growth of the cancercells on a two dimensional surface, such as the skin, lining of the gut,surface of an organ, etc., and selectively illuminating only the cancercells, and not the healthy cells, much more complete killing of thecancer could be achieved without harming the healthy cells.

[0104] Moreover, because multiphoton excitation permits deep probing andscanning of the cells in three dimensions, it has important implicationsfor medical treatments where it is desirable to specifically target andkill some cells while leaving others intact beyond a two dimensionalsurface. For example, the multiphoton excitation technique can be usedto first screen tumor sites in a patient, using a three dimensionallaser scanning methodology (Konig, Journal of Microscopy, Vol. 200, part2, November 2000, pp. 83 -104, hereby incorporated by reference in itsentirety). It can then be used to kill selectively cancer cells whileleaving noncancer cells intact. Similarly, many types of benign skindisorders or cosmetic manipulations, such as the removal of hair cells,could be treated using multiphoton excitation screening and selection.

EXAMPLE 1 Time Correlated Fluorescent Signals from Individual E. coliExpressing Red Fluorescent Protein

[0105]FIG. 8 shows time correlated data taken on a single cell of E.coli expressing red fluorescent protein (RFP) from the dsRED plasmid,available commercially from ClonTech Laboratories, Inc. (Palo Alto,Calif.). The trace was taken using an apparatus similar to the one shownin FIG. 2, but using a sample suspended in solution rather than on asolid substrate.

[0106] Specifically, a frequency doubled, pulse compressed, and modelocked Nd:YAG laser (532 nm, 10 psec) was used to excite the sample at arepetition rate of 82 MHz. To ensure proper beam quality andpolarization, the light was passed through a single mode, polarizationpreserving glass fiber (F-SPA, Newport, Irvine, Calif.) and a polarizingbeam splitter (05BC15PH.3, Newport, Irvine, Calif.). The laser light wasdelivered into an inverted, confocal microscope and reflected up towardsthe microscope objective with a dichroic mirror (Q570LP, Omega Optical,Brattleboro, Vt.).

[0107] The sample, a 50 microliter droplet containing E. coli cellsexpressing the plasmid dsRED to produce red fluorescent protein, wasspread onto a glass cover slip (22×50 mm No 1.5, VWR, West Chester,Pa.). The same objective (100×PlanApo 1.4NA, Olympus, Tokyo Japan) usedto focus the laser also collected the fluorescence.

[0108] The collected fluorescence passed through the dichroic mirror andwas focused onto a 50 micron diameter pinhole (910PH50, Newport, Irvine,Calif.). The fluorescence was then split by a polarizing beam splitter(05FC16PB.3, Newport, Irvine, Calif.), sending photons polarizedparallel to the laser to detector one and photons polarizedperpendicular to the laser to detector two (Perkin Elmer, SPCM-AQR-12,Canada).

[0109] To remove Raleigh and Raman scattering, the fluorescence waspassed through a custom emission filter (Omega Optical, Brattleboro,Vt.). The filter specifically blocks 532 nm light and the water Ramanscattering from 532 nm light. The signal from the detectors and asynchronization signal from each laser pulse were sent into an in-housedesigned and built signal multiplexor/router. The multiplexor sends astart signal and a stop signal into the Timeharp TCSCP board (PicoQuant,Berlin, Germany). The multiplexor also separates the signals frommultiple detectors in regions of time defined by the repetition rate ofthe laser. Each detector's signal occupies a 12 ns region of time.

[0110] The resulting time-correlated fluorescent signals from anindividual E. coli expressing RFP are shown in FIG. 8.

EXAMPLE 2 Light-Mediated Patterning in Cell Selection

[0111] The cationic porphyrin 5,10,15,20-Tetrakis[4-(trimethylammonio)phenyl]-21H,23H-porphine (TmaP), which has amaximum absorption at 412 nm wavelength, was added to a 3 mL liquidculture of E. coli in Luria Broth (LB) and the culture was incubated inthe dark overnight. Approximately 50 uL of the culture was plated on anLB/TmaP agar plate, and portions of the plate was irradiated for about 2minutes with visible light passed through a blue filter. The cells werethen placed in an incubator and allowed to grow overnight at 37° C.

[0112] The image projected onto the plate is shown in FIG. 9A. As shownin FIG. 9B, those portions of the plate receiving visible light (the“white” portion of the image shown in FIG. 9A) show little or no growth.of E. coli, while portions of the plate receiving little or no visiblelight (the “black” portion of the image shown in FIG. 9A) show highdensity growth in the form of the Arizona State University mascot, theSun Devil “Sparky.”

[0113] It is emphasized at this point that the present invention is notintended to be limited to the exemplary embodiments shown and describedabove. The preceding merely illustrates the principles of the invention.It will be appreciated that those skilled in the art will be able todevise various arrangements which, although not explicitly described orshown herein, embody the principles of the invention and are includedwithin its spirit and scope. Furthermore, all examples and conditionallanguage recited herein are principally intended expressly to be onlyfor pedagogical purposes and to aid the reader in understanding theprinciples of the invention and the concepts contributed by theinventors to furthering the art, and are to be construed as beingwithout limitation to such specifically recited examples and conditions.Morever, all statements herein reciting principles, aspects, andembodiments of the invention as well as specific examples thereof, areintended to encompass both structural and functional equivalentsthereof. Additionally, it is intended that such equivalents include bothcurrently known equivalents and equivalents developed in the future,i.e., any elements developed that perform the same function, regardlessof structure. The scope of the present invention, therefore, is notintended to be limited to the exemplary embodiments shown and describedherein. Rather, the scope and spirit of present invention is embodied bythe appended claims.

What is claimed is:
 1. An automated method for analyzing cellscontaining fluorescent markers, the method comprising the steps of:providing a substrate with multiple locations, at least some of whichcontain one or more cells containing a fluorescent marker; directing alight beam onto each location, thereby causing the fluorescent marker toemit fluorescent light; automatically detecting the fluorescent light;automatically measuring and recording the lifetime of the fluorescentlight; and correlating the lifetime of the fluorescent light with thelocation containing the cell with the fluorescent marker emitting thefluorescent light.
 2. The method of claim 1, further comprising the stepof generating an image map of the substrate, the image map indicatingthose locations emitting fluorescent light with a desired lifetime. 3.The method of claim 1, wherein the light beam is directed onto eachlocation through an objective of a scanning microscope.
 4. The method ofclaim 1, wherein the light beam is generated by a high frequency pulsedlaser.
 5. The method of claim 1, wherein the fluorescent light isdetected by an avalanche photodiode.
 6. The method of claim 1, whereinthe lifetime of the fluorescent light is measured by acomputer-interfaced time correlated single photon counting board.
 7. Amethod for imaging and analyzing fluorescence lifetime in cells, themethod comprising the steps of: providing a sample of cells, the cellscontaining a fluorescent marker, wherein the sample is disposed on apositioning stage; scanning a light beam across the sample of cellsthrough an objective of a scanning microscope, causing the cells to emitfluorescent light; detecting the fluorescent light emitted by the cells;measuring the lifetime of the fluorescent light; correlating thelifetime of the fluorescent light with the position of objectiverelative to the sample to thereby generate a high-resolution image mapof cell fluorescence lifetime; and storing the high resolution imagemap.
 8. A method for imaging and analyzing fluorescence lifetime andanisotropy in cells, the method comprising the steps of: providing asample of cells, the cells containing a fluorescent marker, wherein thesample is disposed on a positioning stage; scanning a light beam acrossthe sample of cells through an objective of a scanning microscope,causing the cells to emit fluorescent light; passing the fluorescentlight through a polarizer, to thereby produce two perpendicular planesof polarized light; passing each plane of polarized light through awavelength separator, to thereby produce four fluorescent signals;independently detecting each fluorescent signal; and measuring andrecording the lifetime, wavelength region and polarization of eachfluorescent signal.
 9. The method of claim 8, further comprising thestep of correlating the lifetime, wavelength region and polarization ofeach fluorescent signal with the position of objective relative to thesample to thereby generate a high-resolution image map of cellfluorescence lifetime, wavelength region and polarization.
 10. Anapparatus for detecting spatially imaged fluorescence lifetime,comprising: a high frequency pulsed laser; a light detector; a timecorrelated single photon counting board, the board interfaced with thelaser and with the light detector; and a computer, the computerinterfaced with the time correlated single photon counting board;wherein the laser emits radiation onto a sample thereby causing thesample to emit fluorescent light, the fluorescent light being sensed bythe light detector thereby causing the light detector to generateelectrical pulses, the electrical pulses being sensed by the timecorrelated single photon counting board to thereby measure the lifetimeof the fluorescent light emitted by the sample, and the lifetime of thefluorescent light emitted by the sample being stored in the computer.11. A computer interfaced scanning microscope system for detectingspatially imaged fluorescence lifetime, the system comprising: a highfrequency pulsed laser; an objective lens; a positioner located adjacentto the objective lens, the positioner including a stage designed to holda sample, the positioner designed to move the stage relative to theobjective lens to thereby variably position the sample relative to thefocal point of the objective lens, and the positioner further designedto sense and record the position of the sample relative to the focalpoint of the objective lens as a function of time; a light detector; atime correlated single photon counting board, the board interfaced withthe laser and with the light detector; and a computer, the computerinterfaced with the time correlated single photon counting board;wherein the laser emits a light beam directed through the objectivelens, the objective lens focuses the light beam on the sample on thestage to thereby cause some or all of the molecules in the sample toemit fluorescence; wherein at least a portion of the fluorescence passesthrough the objective to the light detector, the light detectordetecting the fluorescence to thereby generate electrical pulses;wherein the electrical pulses are sensed by the computer-interfaced timecorrelated single photon counting board to thereby determine afluorescence lifetime of the sample, the fluorescence lifetime being theperiod of time between the emission of light by the pulse laser and theemission of the fluorescence from the sample, and to record the time atwhich the fluorescence was detected by the light detector; and whereindata regarding the fluorescence lifetime of the sample and the time atwhich the fluorescence was detected by the light detector are stored inthe computer.
 12. A computer interfaced scanning microscope system fordetecting spatially imaged fluorescence lifetime and anisotropy, thesystem comprising: a high frequency pulsed laser; an objective lens; apositioner located adjacent to the objective lens, the positionerincluding a stage designed to hold a sample, the positioner designed tomove the stage relative to the objective lens to thereby variablyposition the sample relative to the focal point of the objective lens,and the positioner further designed to sense and record the position ofthe sample relative to the focal point of the objective lens as afunction of time; a polarizer; wavelength separators; light detectors;multiplexing electronics interfaced with the light detectors; a timecorrelated single photon counting board, the board interfaced with thelaser and the multiplexing electronics; and a computer, the computerinterfaced with the time correlated single photon counting board;wherein the laser emits a light beam directed through the objectivelens, the objective lens focuses the light beam on the sample on thestage to thereby cause some or all of the molecules in the sample toemit fluorescence; wherein at least a portion of the fluorescence passesthrough the objective to the polarizer, wherein the fluorescence issplit into two planes of polarized light, each of which passes through awavelength separator, the wavelength separator separating each plane ofpolarized light into two fluorescent signals; wherein the lightdetectors detect the fluorescent signals to thereby generate electricalpulses, the electrical pulses being sensed by the computer-interfacedtime correlated single photon counting board to thereby determine thefluorescence lifetime and anisotropy of the sample; and wherein thefluorescence lifetime and anisotropy of the sample are stored in thecomputer.
 13. An automated method for screening and selecting cells, themethod comprising the steps of: providing a substrate with multiplelocations, at least some of which contain one or more cells expressingan imagable property; detecting and recording the imagable property;identifying and recording locations containing cells expressing adesired characteristic of the imagable property and locations notcontaining cells expressing the desired characteristic of the imagableproperty; and scanning lethal irradiation across the substrate through ahigh speed shutter, wherein the shutter is open only when the lethalirradiation is positioned over locations not containing cells expressingthe desired characteristic of the imagable property to thereby kill thecells in such locations.
 14. The method of claim 13, wherein the lethalirradiation is ultraviolet light.
 15. The method of claim 13, whereinthe lethal irradiation is multiphoton excitation of molecules in thecells.
 16. An automated method for screening and selecting cells, themethod comprising the steps of: providing a substrate with multiplelocations, at least some of which contain one or more cells expressingan imagable property; detecting and recording the imagable property;identifying and recording locations containing cells expressing adesired characteristic of the imagable property and locations notcontaining cells expressing the desired characteristic of the imagableproperty; applying a sensitizing agent to the substrate, wherein thesensitizing agent is selected to render the sample of cells sensitive tolight; and scanning a light beam across the substrate through a highspeed shutter, wherein the shutter is open only when the light beam ispositioned over locations not containing cells expressing the desiredcharacteristic of the imagable property to thereby kill the cells insuch locations.
 17. The method of claim 16 wherein the sensitizing agentis a DNA intercalating dye.
 18. The method of claim 17 wherein the DNAintercalating dye is ethidium bromide.
 19. The method of claim 16wherein the sensitizing agent is a porphyrin.
 20. The method of claim 16wherein the sensitizing agent generates reactive oxygen species uponabsorption of light.
 21. An automated method for screening and selectingcells based on fluorescent amplitude, the method comprising the stepsof: providing a substrate with multiple locations, at least some ofwhich contain one or more cells containing a fluorescent marker;directing a light beam onto each location, thereby causing thefluorescent marker to emit fluorescent light; automatically detectingthe fluorescent light; automatically measuring and recording theamplitude of the fluorescent light; correlating the amplitude of thefluorescent light with the location containing the cell with thefluorescent marker emitting the fluorescent light; generating a kill mapof the substrate, the kill map indicating those locations not emittingfluorescent light with the desired amplitude; and scanning lethalirradiation across the substrate through a high speed shutter, whereinthe shutter is open only when the lethal irradiation is positioned overlocations not emitting fluorescent light with the desired amplitude tothereby kill the cells in such locations.
 22. The method of claim 21,wherein the lethal irradiation is ultraviolet light.
 23. The method ofclaim 21, wherein the lethal irradiation is multiphoton excitation ofmolecules in the cells.
 24. An automated method for screening andselecting cells based on fluorescent amplitude, the method comprisingthe steps of: providing a substrate with multiple locations, at leastsome of which contain one or more cells containing a fluorescent marker;directing a light beam onto each location, thereby causing thefluorescent marker to emit fluorescent light; automatically detectingthe fluorescent light; automatically measuring and recording theamplitude of the fluorescent light; comparing the amplitude of thefluorescent light to a predetermined desirable fluorescent amplitude, tothereby determine whether the location contains a cell emittingfluorescent light with the desired amplitude; and directing lethalirradiation to those locations that do not contain a cell emittingfluorescent light with the desired amplitude to thereby kill the cellsin such locations.
 25. The method of claim 24, wherein the lethalirradiation is ultraviolet light.
 26. The method of claim 24, whereinthe lethal irradiation is multiphoton excitation of molecules in thecells.
 27. An automated method for screening and selecting cells basedon fluorescent amplitude, the method comprising the steps of: providinga substrate with multiple locations, at least some of which contain oneor more cells containing a fluorescent marker; directing a light beamonto each location, thereby causing the fluorescent marker to emitfluorescent light; automatically detecting the fluorescent light;automatically measuring and recording the amplitude of the fluorescentlight; correlating the amplitude of the fluorescent light with thelocation containing the cell with the fluorescent marker emitting thefluorescent light; generating a kill map of the substrate, the kill mapindicating those locations not emitting fluorescent light with thedesired amplitude; applying a sensitizing agent to the substrate,wherein the sensitizing agent is selected to render the sample of cellssensitive to light; and scanning a light beam across the substratethrough a high speed shutter, wherein the shutter is open only when thelight beam is positioned over locations not emitting fluorescent lightwith the desired amplitude to thereby kill the cells in such locations.28. The method of claim 27 wherein the sensitizing agent is a DNAintercalating dye.
 29. The method of claim 28 wherein the DNAintercalating dye is ethidium bromide.
 30. The method of claim 27wherein the sensitizing agent is a porphyrin.
 31. The method of claim 27wherein the sensitizing agent generates reactive oxygen species uponabsorption of light.
 32. An automated method for screening and selectingcells based on fluorescent amplitude, the method comprising the stepsof: providing a substrate with multiple locations, at least some ofwhich contain one or more cells containing a fluorescent marker;directing a light beam onto each location, thereby causing thefluorescent marker to emit fluorescent light; automatically detectingthe fluorescent light; automatically measuring and recording theamplitude of the fluorescent light; correlating the amplitude of thefluorescent light with the location containing the cell with thefluorescent marker emitting the fluorescent light; generating a kill mapof the substrate, the kill map indicating those locations not emittingfluorescent light with the desired amplitude; inducing the cells tosynthesize an endogenous porphyrin precursor; and scanning a light beamacross the substrate through a high speed shutter, wherein the shutteris open only when the light beam is positioned over locations notemitting fluorescent light with the desired amplitude to thereby killthe cells in such locations.
 33. An automated method for screening andselecting cells based on fluorescent lifetime, the method comprising thesteps of: providing a substrate with multiple locations, at least someof which contain one or more cells containing a fluorescent marker;directing a light beam onto each location, thereby causing thefluorescent marker to emit fluorescent light; automatically detectingthe fluorescent light; automatically measuring and recording thelifetime of the fluorescent light; correlating the lifetime of thefluorescent light with the location containing the cell with thefluorescent marker emitting the fluorescent light; generating a kill mapof the substrate, the kill map indicating those locations not emittingfluorescent light with the desired lifetime; and scanning lethalirradiation across the substrate through a high speed shutter, whereinthe shutter is open only when the lethal irradiation is positioned overlocations not emitting fluorescent light with the desired lifetime tothereby kill the cells in such locations.
 34. The method of claim 33,wherein the lethal irradiation is ultraviolet light.
 35. The method ofclaim 33, wherein the lethal irradiation is multiphoton excitation ofmolecules in the cells.
 36. An automated method for screening andselecting cells based on fluorescent lifetime, the method comprising thesteps of: providing a substrate with multiple locations, at least someof which contain one or more cells containing a fluorescent marker;directing a light beam onto each location, thereby causing thefluorescent marker to emit fluorescent light; automatically detectingthe fluorescent light; automatically measuring and recording thelifetime of the fluorescent light; comparing the lifetime of thefluorescent light to a predetermined desirable fluorescent lifetime, tothereby determine whether the location contains a cell emittingfluorescent light with the desired lifetime; and directing lethalirradiation to those locations that do not contain a cell emittingfluorescent light with the desired lifetime to thereby kill the cells insuch locations.
 37. The method of claim 36, wherein the lethalirradiation is ultraviolet light.
 38. The method of claim 36, whereinthe lethal irradiation is multiphoton excitation of molecules in thecells.
 39. An automated method for screening and selecting cells basedon fluorescent lifetime, the method comprising the steps of: providing asubstrate with multiple locations, at least some of which contain one ormore cells containing a fluorescent marker; directing a light beam ontoeach location, thereby causing the fluorescent marker to emitfluorescent light; automatically detecting the fluorescent light;automatically measuring and recording the lifetime of the fluorescentlight; correlating the lifetime of the fluorescent light with thelocation containing the cell with the fluorescent marker emitting thefluorescent light; generating a kill map of the substrate, the kill mapindicating those locations not emitting fluorescent light with thedesired lifetime; applying a sensitizing agent to the substrate, whereinthe sensitizing agent is selected to render the sample of cellssensitive to light; and scanning a light beam across the substratethrough a high speed shutter, wherein the shutter is open only when thelight beam is positioned over locations not emitting fluorescent lightwith the desired lifetime to thereby kill the cells in such locations.40. The method of claim 39 wherein the sensitizing agent is a DNAintercalating dye.
 41. The method of claim 39 wherein the sensitizingagent is a porphyrin.
 42. The method of claim 39 wherein the sensitizingagent generates a reactive oxygen species upon absorption of light. 43.An automated method for screening and selecting cells based onfluorescent lifetime, the method comprising the steps of: providing asubstrate with multiple locations, at least some of which contain one ormore cells containing a fluorescent marker; directing a light beam ontoeach location, thereby causing the fluorescent marker to emitfluorescent light; automatically detecting the fluorescent light;automatically measuring and recording the lifetime of the fluorescentlight; correlating the lifetime of the fluorescent light with thelocation containing the cell with the fluorescent marker emitting thefluorescent light; generating a kill map of the substrate, the kill mapindicating those locations not emitting fluorescent light with thedesired lifetime; inducing the cells to synthesize an endogenousporphyrin precursor; and scanning a light beam across the substratethrough a high speed shutter, wherein the shutter is open only when thelight beam is positioned over locations not emitting fluorescent lightwith the desired lifetime to thereby kill the cells in such locations.44. An automated method for screening and selecting cells, the methodcomprising the steps of: providing a substrate with multiple locations,at least some of which contain one or more cells expressing an imagableproperty; detecting and recording the imagable property; identifying andrecording locations containing cells expressing a desired characteristicof the imagable property and locations not containing cells expressingthe desired characteristic of the imagable property; and projectinglethal irradiation only onto those locations not containing cellsexpressing the desired characteristic of the imagable property tothereby selectively kill those cells.
 45. The method of claim 44,wherein a computer-controlled projection device is used to project thelethal irradiation onto locations not containing cells expressing thedesired characteristic of the imagable property.
 46. The method of claim44, wherein the lethal irradiation is ultraviolet light.
 47. The methodof claim 44, wherein the lethal irradiation is multiphoton excitation ofmolecules in the cells.
 48. The method of claim 44, wherein the imagableproperty of the cells is sensed and recorded by a charge couple devicebased (CCD) camera.
 49. An automated method for screening and selectingcells, the method comprising the steps of: providing a substrate withmultiple locations, at least some of which contain one or more cellsexpressing an imagable property; detecting and recording the imagableproperty; identifying and recording locations containing cellsexpressing a desired characteristic of the imagable property andlocations not containing cells expressing the desired characteristic ofthe imagable property; applying a sensitizing agent to the cells,wherein the sensitizing agent is selected to render the cells sensitiveto light; and projecting light only onto those locations not containingcells expressing the desired characteristic of the imagable property tothereby selectively kill those cells.
 50. The method of claim 49 whereinthe sensitizing agent is a DNA intercalating dye.
 51. The method ofclaim 50, wherein the DNA intercalating dye is ethidium bromide.
 52. Themethod of claim 49 wherein the sensitizing agent is a porphyrin.
 53. Themethod of claim 49 wherein the sensitizing agent generates a reactiveoxygen species upon absorption of light.
 54. The method of claim 49,wherein the imagable property of the cells is sensed and recorded by acharge couple device based (CCD) camera.
 55. The method of claim 49,wherein a computer-controlled projection device is used to project thelight onto the cells not exhibiting the desired characteristic of theimagable property.
 56. The method of claim 49, wherein thecomputer-controlled projection device is a digital light processor. 57.A method for selecting cells exhibiting desirable fluorescenceproperties, the method comprising the steps of: providing a sample ofcells, wherein the cells contain fluorescent markers that emitfluorescent light when excited; exciting the sample of cells; sensingand recording the fluorescence properties of the fluorescent lightemitted by the cells in the sample; generating a high resolution imagemap of the cells, based on the fluorescence properties of the cells,indicating those cells exhibiting desirable fluorescent properties aswell as those cells not exhibiting desirable fluorescent properties; andprojecting lethal irradiation only onto those cells not exhibitingdesirable fluorescence properties to thereby selectively kill thosecells.
 58. The method of claim 57, wherein a computer-controlledprojection device is used to project the lethal irradiation onto thecells not exhibiting desirable fluorescence properties.
 59. The methodof claim 57, wherein the lethal irradiation is ultraviolet light. 60.The method of claim 57, wherein the lethal irradiation is multiphotonexcitation of molecules in the cells.
 61. The method of claim 57,wherein the fluorescence properties of the cells are sensed and recordedby a charge couple device based (CCD) camera.
 62. A method for selectingcells exhibiting desirable fluorescence properties, the methodcomprising the steps of: providing a sample of cells, wherein the cellscontain fluorescent markers that emit fluorescent light when excited;exciting the sample of cells; sensing and recording the fluorescenceproperties of the fluorescent light emitted by the cells in the sample;generating a high resolution image map of the cells, based on thefluorescence properties of the cells, indicating those cells exhibitingdesirable fluorescent properties as well as those cells not exhibitingdesirable fluorescent properties; applying a sensitizing agent to thesample of cells, wherein the sensitizing agent is selected to render thesample of cells sensitive to light; and projecting light only onto thosecells not exhibiting desirable fluorescence properties to therebyselectively kill those cells.
 63. The method of claim 62 wherein thesensitizing agent is a DNA intercalating dye.
 64. The method of claim 62wherein the sensitizing agent is a porphyrin.
 65. The method of claim 62wherein the sensitizing agent generates a reactive oxygen species uponabsorption of light.
 66. The method of claim 62, wherein thefluorescence properties of the cells are sensed and recorded by a chargecouple device based (CCD) camera.
 67. The method of claim 62, wherein acomputer-controlled projection device is used to project the light ontothe cells not exhibiting desirable fluorescent properties.
 68. Themethod of claim 67, wherein the computer-controlled projection device isa digital light processor.
 69. A method for selecting cells exhibitingdesirable fluorescence properties, the method comprising the steps of:providing a sample of cells, wherein the cells contain fluorescentmarkers that emit fluorescent light when excited; exciting the sample ofcells; sensing and recording the fluorescence properties of thefluorescent light emitted by the cells in the sample; generating a highresolution image map of the cells, based on the fluorescence propertiesof the cells, indicating those cells exhibiting desirable fluorescentproperties as well as those cells not exhibiting desirable fluorescentproperties; inducing the cells to synthesize an endogenous porphyrinprecursor; and projecting light only onto those cells not exhibitingdesirable fluorescence properties to thereby selectively kill thosecells.
 70. The method of claim 69, wherein the fluorescence propertiesof the cells are sensed and recorded by a charge couple device based(CCD) camera.
 71. The method of claim 69, wherein a computer-controlledprojection device is used to project the light onto the cells notexhibiting desirable fluorescent properties.
 72. The method of claim 71,wherein the computer-controlled projection device is a digital lightprocessor.
 73. A method for selectively killing cells, the steps of themethod comprising: applying a diagnostic fluorophore to a population ofcells, wherein some cells in the population show identifiablefluorescent characteristics compared to other cells in the population;scanning the population of cells with a light beam from an ultrafastlaser, the light beam having a wavelength and intensity selected todetect multiphoton excitation of the fluorophore without causingsubstantial cell death, wherein the fluorophore in the cells is excitedby multiphoton absorption at a focal point of the light beam, causingthe fluorophore to emit fluorescent light; detecting the fluorescentlight; generating a high resolution kill map of the population of cells,the kill map indicating those cells emitting fluorescent light inresponse to the light beam; scanning the population of cells with a highintensity light beam from the ultrafast laser, the high intensity lightbeam having a wavelength and intensity selected to kill the cells,through a high speed shutter, wherein the shutter is open only when thehigh intensity light beam is focused on cells emitting fluorescentlight, to thereby selectively kill those cells.
 74. The method of claim73, wherein the diagnostic fluorophore is a porphyrin.
 75. A method forselectively killing cells, the steps of the method comprising: applyinga diagnostic fluorophore to a population of cells, wherein some cells inthe population selectively absorb the fluorophore at a higher rate thanother cells in the population to thereby emit more fluorescent lightwhen excited relative to the other cells; focusing a light beam from anultrafast laser on the population of cells, the light beam having awavelength and intensity selected to detect multiphoton excitation ofthe fluorophore without causing substantial cell death, wherein thefluorophore in the cells is excited by multiphoton absorption at thefocal point of the light beam, causing the fluorophore to emitfluorescent light; detecting the fluorescent light and measuring thetotal quantity of fluorescent light emitted at the focal point of thelight beam; comparing the total quantity of fluorescent light emitted atthe focal point of the light beam to a pre-determined total quantity offluorescent light emitted by cells not selectively absorbing thefluorophore to thereby determine whether the cells at the focal point ofthe light beam have selectively absorbed the fluorophore at a higherrate; and increasing the intensity of the light beam from the ultrafastlight beam to thereby selectively kill only those cells that haveselectively absorbed the fluorophore.
 76. The method of claim 75,wherein the diagnostic fluorophore is a porphyrin